This invention relates to biologically active xcex2-glucan-mannan preparations and to methods for their isolation. In particular, the invention relates to xcex2-glucan-mannan preparations including xcex2 (1-3) glucan, produced from microorganisms including but not limited to yeasts, and to methods for producing the xcex2-glucan-mannan preparations which avoid the use of concentrated alkali or acid.
Polysaccharides are widely distributed in nature, and are particularly important for their role in maintaining the structural integrity of bacterial fungal, and plant cells. Glucans are polymers of D-glucose, and the D-glucose units may be linked together in a variety of ways. For example, glucans with 1-3, 1-4, 1-6, and 1-2 linkages are all known. The variety of linkages possible means that glucans are normally highly branched compounds. Because of their chemical properties, glucans have found a wide variety of uses in the chemical, food and pharmaceutical industries. For example, they are useful as viscosity imparting agents, emulsifiers, fibers, films, coating substances, supports for affinity chromatography and gel electrophoresis, in cell culture media, as filter pads, and in cement. They are also widely used as food thickeners and as a source of dietary fibre, and as carriers and coating agents in pharmaceutical products.
Glucans, particularly xcex2 (1-3)-glucans, have been very extensively studied, and in addition to the foregoing, have been shown to have a variety of pharmacological activities, including but not limited to anti-cholesterolaemic activity, hypoglycaemic activity, acceleration of heavy metal excretion, and stimulation of the immune system. The immunostimulatory activity of xcex2-glucans has led to suggestions that they are useful as anti-cancer agents or in the treatment of HIV infection, as agents for stimulation of wound healing, or as anti-infective agents for use either alone or in conjunction with antibiotics.
The xcex2-glucans can also induce the resistance response to disease in plants, such as phytoalexin production and wilting. This has led to suggestions that they can be used as anti-infective agents and growth promoters in plants.
Because of the problems entailed in intensive poultry, animal, fish or crustacean production, the use of xcex2-glucans as an additive to these feeds, to reduce incidence of infection and consequently to promote growth and to reduce the need for antibiotics, has also been proposed.
xcex2 (1-3)-glucan is an important cell wall component of yeast cells. The cell wall of Saccharaomyces cerevisiae is primarily composed of xcex2-linked glucan, which is mainly a backbone of xcex2 (1-3)-linked glucose units, with a minor component of inter and intra molecular branching via xcex2 (1-6)-linkages. Because of the very wide use of yeasts in the food and brewing industry, as well as in the production of industrial-grade alcohol, spent yeast cells are a major industrial by-product. Yeast-derived products themselves have considerable. commercial value, for example in such products as yeast extracts, flavouring agents, flavour potentiators such as guanosine monophosphate and inosine monophosphate, and in the manufacture of enzymes, fine chemicals and products for use in the biochemical and pharmaceutical industries, such as trehalose, thymidine, nucleosides and nucleotides, etc. Waste yeast from the brewing industry is a major source of xcex2-glucans.
In addition, other species of yeast are also useful as a source of xcex2-glucans, including but not limited to other yeast strains of Saccharomyces cerevisiae, Kluyveromyces fragilis, and Candida strains such as Candida utilis. All of these yeast strains can be produced using culture in food grade nutrients either by batch fermentation or continuous fermentation. Many other species of microorganisms, including bacteria, fungi and unicellular algae, have been reported in the art as a source of xcex2-glucans.
The purification of xcex2-glucans from yeast and other organisms has been extensively investigated, and a variety of methods is known. Most of these rely on the insolubility of xcex2 (1-3)-glucan in alkali or in organic solvents. The principal known methods are:
(a) High temperature extraction with concentrated sodium hydroxide, followed by high temperature extraction with acid and precipitation with ethanol (see for example Manners, D. J. et al., Biochem. J. 135 19-30 (1973), Jamas, S. et al., U.S. Pat. Nos. 4,810,646, No. 5,028,703, and No. 5,250,436 and Australian Patent No. 628752. by Phillips Petroleum Company). Many of these protocols require preliminary homogenisation of the yeast cells, and many require multiple repetition of each extraction step.
(b) Extraction with concentrated sodium hydroxide, followed by high temperature acid extraction and enzyme treatment to modify or purify the glucan (see for example Czech Patent Application No. 890038 by Masler, L. et al. which reports purification of xcex2-D-glucan by alkali-acid extraction, followed by treatment with enzymes having amylase activity).
(c) Extraction of yeast cell wall preparations resulting from autolysis or enzyme degradation of yeast with concentrated phenol: water (1:1) (see for example U.S. Pat. No. 4,138,479 by Truscheit, E. et al.).
(d) Extraction with organic solvents such as isopropanol, ethanol, acetone, or methanol either alone or in the presence of alkali (see for example Japanese Patent publications No. 7051081, 6340701, 5295003, and 3002202; European Patent Application No. 515216).
Acid treatment is known to reduce the number of xcex2 (1-6)-linkages in the glucan material and this results in an increase in viscosity.
The cell wall of yeast is mainly composed of:
(i) fibrillar, alkali insoluble xcex2 (1-3)-linked glucan, with side branches of xcex2 (1-6)-linked glucan.
(ii) alkali-soluble xcex2 (1-3)-linked glucan with side branches of xcex2 (1-6)-linked glucan.
(iii) amorphous acid-soluble xcex2 (1-6)-glucan, with intermittent xcex2 (1-3)-linkages.
(iv) amorphous alkali-soluble mannan linked to proteins.
The fibrillar glucan component is located adjacent to the yeast plasma membrane, and is covered externally by an amorphous layer composed mainly of mannoproteins (Kopecka M. et al., J. Cell Biol., 62 68-76 (1974)). Particulate material (zymosan) isolated from the cell walls of Saccharomyces cerevisiae is known to have the ability to act as a non-specific immune stimulant. The biological activity of yeast cell wall particulates is largely attributed to the presence of xcex2 (1-3)-linked glucan, but the other two forms of glucan and mannan also have some ability to stimulate the immune system. Mannan is reported to mediate the adsorption and phagocytosis of particulate material, such as insoluble xcex2 (1-3)-glucan, by cells of the immune system (Giaimis, J., et al., Journal Of Leukocyte Biology 54 564-571 (1993); Sun-Sang, J., et al., Journal Of Cell Biology 96 160-166 (1983)).
Existing methods to isolate the xcex2-glucans commonly use a multi-step alkali-acid extraction process (Manners, D. J., et al., J. Gen. Micro. 80 411-417 (1974)). The alkali extraction steps remove most of the amorphous mannoprotein and glucan material, and the subsequent acid extraction steps remove the glycogen and most of the xcex2 (1-6)-side branches from the fibrillar predominantly xcex2 (1-3) linked glucan. A final solvent extraction step is sometimes used to remove lipids.
It is clear that, given the retail price of glucan for some applications, the cost of producing glucan using existing published or patented methods is not commercially viable. These methods have the following problems:
(i) They are aimed at only producing the fibrillar, alkali-insoluble form of glucan. Other forms of glucan and the mannan present in the cell wall are removed as by-products of the process. These represent an additional amount of glucan, which could have significantly increased the glucan yield, and which may be functionally important.
(ii) The existing processes require significant capital investments.
(iii) The existing processes are hazardous, because of the requirement for high concentration caustic and acid treatments at high temperatures.
(iv) The processes result in a solution containing an alkali-insoluble glucan, which is difficult to separate based on conventional techniques, and this results in poor recoveries.
(v) The cost of production is high when compared to the value of the products in many applications.
Electron microscope studies of yeast cell walls (Kopecka M., et al., J. Cell Biol., 62 68-76 1974). showed that the fibrillar component of the cell wall was revealed when the outer amorphous layer of mannoproteins was removed by treatment with enzymes.
There is a clear need in the art for a rapid and inexpensive method of xcex2-glucan extraction which avoids the use of high concentrations of alkali or acid and the use of high temperatures, which avoids loss of alkali soluble glucans and mannans, which has improved recovery of glucans and mannans and which results in a biologically active preparation.
We have now surprisingly found that a xcex2-glucan-mannan preparation can be isolated using a simple autolysis process, at near-neutral pH and only slightly elevated temperature, and that excellent yields of a product with high immunostimulatory activity which may include stimulation of phagocytic activity, superoxide production, increased resistance to pathogens and infections, are obtained. Autolysis may be supplemented by treatment under gentle conditions with enzymes or other agents and if desired, the properties of the glucan-mannan product can be modified by acid treatment, degree of homogenisation or by varying the type of enzyme used.